Scatchard analysis of fluorescent concanavalin a binding to lymphocytes

Abstract

Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothiocyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4°C was compared to ¹²⁵I-ConA binding. A FACS IV flow cytometer (Becton-Dickinson, Mountain View, CA) was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer-determined fluorescence and ligand binding per cell. As FITC-ConA binding showed a quasi-Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC-ConA binding yielded results (1.9 × 10⁶ receptors/cell, K = 3.6 × 10⁻¹⁵) similar to those obtained with ¹²⁵I-ConA (1.4 × 10⁶ receptors/cell, K = 5.2 × 10⁻¹⁵). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations.